WO2001040499A2 - Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications - Google Patents
Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications Download PDFInfo
- Publication number
- WO2001040499A2 WO2001040499A2 PCT/FR2000/003342 FR0003342W WO0140499A2 WO 2001040499 A2 WO2001040499 A2 WO 2001040499A2 FR 0003342 W FR0003342 W FR 0003342W WO 0140499 A2 WO0140499 A2 WO 0140499A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- sequence
- plasmid
- promoter
- yeast
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Definitions
- the present invention relates to methods of cell screening for compounds capable of modulating the activity of ubiquitin-SCF ligase complexes and their applications.
- the proteasome is a protein complex, composed of several subunits, which recognizes proteins when they have been modified by the attachment of ubiquitin to their lysine residues.
- This ubiquitinylation prior to the recognition of target proteins by the proteasome, involves at least three enzyme complexes, called E1, E2 and E3. It catalyzes the activation of ubiquitin by forming a thioester between itself and ubiquitin, which is then transferred to the enzyme E2, the conjugation enzyme.
- E1 E2 enzyme complexes
- E3 ligase facilitates the recognition of the target by E2 or directly transfers the ubiquitin from E2 onto the substrate (Hochstrasser M. et al., (1996), Annu. Rev. Genêt, 30, 405-439).
- E4 an additional conjugation factor
- E3 proteins in particular the family of HECT proteins (“Homologous to E6-AP Carboxyl-Terminus”) which have a carboxy-terminal domain homologous to that of the human E6-AP protein, involved in the formation of a catalytic intermediate with ubiquitin, and - E3 complexes, among which the family of ubiquitin-SCF ligase complexes (S: Skpl; C: Cdc53 or culline; F: protein containing an F box), is the most diverse. SCF ligases were discovered in the yeast Saccharomyces cerevisiae (Feldman RMR et al., (1997), Cell, 91, 221-230; Skowyra D.
- the SCF complexes comprise at least three common subunits, the Skpl protein, a protein of the culline family (Cdc53 in yeast and culline 1 in humans) and the Hrtl protein (Rbxl or Roxl). They also include modular receptor subunits, which confer specificity with respect to the substrate, and which are proteins containing an F box (Skowyra D.
- the F domain is a degenerate motif of approximately 40 amino acids, which allows the protein which contains it, to interact specifically with Skpl (Bai C. et al, (1996), Cell, 86, 263-274).
- the SCF complexes are very closely associated with a particular E2 enzyme, called Cdc34, which recognizes an independent binding site on Cdc53 (Patton EE et al, (1998), Genes dev cited reference).
- SCF cdc4 targets one or more specific substrates which will be degraded after ubiquitinylation; thus SCF Cdc4 targets CDK inhibitors (cyclin-dependent kinases), Siclp and Farlp, SCF Grrl targets cyclins Gl, Clnl / Cln2 and SCF Met3 ° targets CDK inhibitor, Swepl (Koepp DM et al, (1999), cited reference).
- the inventors have shown that the SCF Met30 complex also plays a role in the downregulation of the metabolism of sulfur amino acids (Thomas D. et al, (1995), Mol. Cell. Biol, 15, 6526-6534).
- SCF Met30 complex Counterparts of the SCF Met30 complex have recently been discovered in other organisms.
- a Slimb gene (Supernumerary Limbs) encoding F-box proteins has been identified, which target the inhibitory protein I ⁇ B, the transcriptional co-activator ⁇ -catenin / Armadillo ( ⁇ -cat / Arm) or the regulatory protein Cubitus interruptus (Ci) (Spencer E. et al, (1999), Genes. Dev., 13, 284-294).
- the protein ⁇ -TrCP ( ⁇ -transducing repeat containing protein) has been described, for the first time, in the context of infection with the NIH-1 virus. It is an F-box protein, induced by the viral protein Vpu, (Margottin F. et al, (1998), Mol. Cell, 1, 565-574), which is involved in the degradation of CD4 cell receptors present on the surface of infected cells, and is necessary to obtain infectious HIV viral particles.
- ⁇ -TrCP allows specific recognition and destruction of targets such as I ⁇ B ⁇ , an inhibitor of the ubiquitous transcription factor ⁇ FKB, which directly regulates the immune and inflammatory response, ⁇ -catenin, protein of the Wg / Wnt pathway , whose abnormal stabilization leads to activa tion of transcription of oncogenic genes and which is involved in several types of cancer (Hart M. et al, (1999), Curr. Biol, 9, 207-210; Kroll et al, (1999), J. Biol Chem., 274, 7941-7945). More recently, a protein homologous to ⁇ -TrCP, the protein FWD1, has been identified in mice (Hatakeyama S. et al., (1999), Proc. Natl. Acad. Sci. USA, 96, 3859-3863).
- the SCF complexes in fact constitute the prototypes of an even larger super family of ubiquitin ligases, also comprising the APC (anaphase promoting complex) complexes, which control cell division, and the VCB complexes (VHL-ElonginC / ElonginB), in particular those which intervene in certain rare forms of hereditary cancers and those which contain a SOCS box (suppr essor ofcytokine signaling).
- APC anaphase promoting complex
- VHL-ElonginC / ElonginB VCB complexes
- SOCS box uppr essor ofcytokine signaling
- the inventors have thus identified, in yeast, the protein Met30 as a factor involved in the transcriptional repression of the genes involved in the biosynthesis of sulfur amino acids (cysteine, methionine and S-adenosylmethionine (AdoMet)) (Thomas D. et a , (1995), Mol. Cell. Biol, 15, 6526-6534).
- this metabolic pathway has a set of about 25 genes (genes).
- the Cbfl-Met4-Met28 complex binds to the TCACGTC sequence upstream of the METX6 gene while the Met4-Met28-Met31 and Met4-Met28-Met32 complexes recognize the AAACTGTG motif upstream of the MET3 and MET28 genes (Kuras L. and al (1997), EMBO J., 16, 2441-2451; Blaiseau PL et al. (1998), EMBO J., 17.6327-6336).
- the transcriptional activation of the different MET genes depends only on a single activation domain carried by the Met4 subunit.
- the dysfunction of the ubiquitin-proteasome pathway has been established in many pathologies of extremely diverse natures, notably cancers, genetic diseases, Parkinson's disease, Alzheimer's disease, inflammatory syndromes and viral infections.
- any mutation present on the target proteins around the phosphorylation sites abolishes the recognition of the mutated proteins by the SCF complexes and leads to their stabilization.
- mutations which affect ⁇ -catenin and prohibit its destruction are involved in tumor transformation in many tissues (colon cancer, melanoma, hepatocarcinoma, etc.).
- the excessive degradation of ⁇ -catenin in neurons has been implicated in Alzheimer's disease, and is implicated in neuronal death by apoptosis which occurs in this pathology.
- - PCT International Application WO 97/12962 describes, for screening anticancer agents, a process which implements an ubiquitin ligase E3 containing a C-terminal region homologous to the catalytic domain of the E6-AP protein (family "HECT"): h-publ, h-pub2, h-pub3, s-publ) capable of specifically ubiquitinylating a cell cycle regulatory protein such as tyrosine phosphatase cdc 25 or the tumor suppressor p53, - PCT International Application WO 99/04033 and US Pat. No.
- 5,932,425 for screening substances capable of treating disorders characterized by an increase in the transcriptional activity of factor NF- ⁇ B (autoimmune diseases, inflammatory conditions, cachexia, AIDS), describe a process which also implements a human ubiquitin ligase of the HECT family (RSC or KIAAN), capable of specifically ubiquitinylating the I ⁇ B protein which forms an inactive cytoplasmic complex with the transcrip factor ubiquitous NF- ⁇ B, which regulates the immune and inflammatory response,
- RSC or KIAAN human ubiquitin ligase of the HECT family
- the inventors Taking advantage of the extreme conservation of the molecular mechanisms involved in the ubiquitin-proteasome pathway of protein degradation in all eukaryotic cells, the inventors have developed a cell screening process for compounds capable of acting on pathologies linked to the dysfunction of the ubiquitin-proteasome cascade in humans using this route in a way that had never been envisaged. They have, in fact, selected a protein from the ubiquitin-proteasome pathway, the stability of which is not directly implicated in the pathologies to be treated, but which provides very efficient screening tools, which have the advantage of be fast and reliable.
- the present invention therefore relates to methods of cell screening for compounds or agents capable of modulating the activity of SCF Met30 complexes, characterized in that they comprise the following steps:
- a modified yeast strain comprising (a) a hybrid sequence, comprising a sequence coding for a Met4 protein, in its wild or mutated form, fused in phase with at least one sequence coding for a suitable marker, said hybrid sequence being expressed under the control of a promoter active in yeast and optionally (b) a reporter transcription system consisting of a reporter gene placed under the control of a suitable operator sequence or an appropriate yeast promoter, (ii) the addition of methionine, at repressive concentrations, between 0.03 mM and 20 mM, or non-repressive, and (iii) determining the level of expression and the stability of the protein expressed from the hybrid sequence, either by visualization and / or quantification, or by determination of the activity of the reporter gene.
- said methods can comprise in parallel the following steps:
- a modified yeast strain comprising a hybrid sequence, comprising a sequence coding for a Met30 protein, in its wild or mutated form, fused in phase with at least one sequence coding for a appropriate marker, said hybrid sequence being expressed under the control of a promoter, active in yeast,
- said methods can also comprise, an additional means implementing a cellular control system, which will make it possible to ensure the specificity of response of the systems hybrids or transcriptional reporter systems used in steps (i) to (vi), said means comprising the following steps:
- a modified yeast strain comprising a hybrid sequence, comprising a sequence coding for a Met28 protein, in its wild or mutated form, fused in phase with at least one sequence coding for a appropriate marker, said hybrid sequence being expressed under the control of a promoter, active in yeast,
- said methods can also comprise, an additional means implementing a cell-free control system which is based on measurement transcription rates of the hybrid sequences and of the metabolic genes MET 16 and MET 25, said means comprising the following steps:
- step (x) the extraction of total RNAs from the modified strains used either in step (i), or in step (iv), or in step (vii) and (xi) measurement of the transcription rates of the hybrid sequence and that of the metabolic genes MET 16 and MET25.
- a subject of the present invention is also methods of cell screening for compounds capable of modulating the activity of SCF Met3 ° complexes, characterized in that they comprise the following steps: (xii) bringing the product to be tested into contact with a modified yeast strain, comprising a reporter transcription system, consisting of a reporter gene placed under the control of an appropriate yeast promoter,
- mutated form of a protein means a protein modified either by insertion, deletion or substitution of one or more amino acids.
- methionine is intended to mean the form (L) or the form (DL) of methionine.
- the term “operator sequence or appropriate operator controlling the reporter gene” means a sequence recognized by a DNA binding factor fused in phase with a Met4 protein, such as the LexA fusion protein. -Met4.
- the term “yeast promoter controlling the reporter gene” means a promoter activated by the transcription factor Met4, such as a MET gene such as MET3, MET10, MET14, MET16, MET25 or MET28.
- the marker used for the construction of the hybrid sequence is chosen from the group consisting of: antigenic peptides, for example the hemagglutinin (Ha) antigenic motif, intrinsically fluorescent proteins, like by example the Green Fluorescence Protein (GFP), proteins with measurable enzymatic activity, and DNA binding factors such as for example LexA from E. coli.
- antigenic peptides for example the hemagglutinin (Ha) antigenic motif
- intrinsically fluorescent proteins like by example the Green Fluorescence Protein (GFP)
- GFP Green Fluorescence Protein
- the promoter allowing the expression of the hybrid protein can be either an inducible promoter active in S. cerevisiae, which can be advantageously chosen from the group constituted by the promoter of the GAL1 gene and the promoter of the CUP1 gene, ie a constitutive promoter, such as for example the promoter of the ADH1 gene of S. cerevisiae.
- the reporter gene is chosen from the group consisting of reporter genes whose activity can be viewed by a colorimetric method, such as for example the XylE gene from E. putida and the LacZ gene of. coli, and metabolic genes, the activity of which can be measured by a growth test, in particular the HIS3, URA3, TRP1 and LEU2 genes of S. cerevisiae.
- Said metabolic genes can alternatively and advantageously be used as selection genes for modified yeasts comprising a hybrid sequence and / or a reporter transcription system, as defined above.
- said reporter gene present in the reporter transcription system
- LexA-Met4 (LexA operators).
- the yeast strains carry one or more mutations which increase the permeability to the products to be tested (Vidal M et al, (1999), Trends in Biotechnol, 17, 374 -381).
- yeast strains having the genetic background of the strain W303 of yeast S. cerevisiae which is described by Bailis AM et al, (Genetics, (1990), 126, 535-547) or any other strain characterized by said yeast.
- the yeasts can be transformed by plasmids constructed by conventional molecular biology techniques, in particular according to the protocols described by Sambrook J. et al. (Molecular cloning Laboratory Manual, 2 nd edition, (1989), Cold Spring Harbor, NY) and Ausubel FM et al. (Current Protocols in Molecular Biology, (1990-1999), John Wiley and Sons, Inc. New York).
- the activity of the reporter genes and of the MET genes is measured, according to the reporter gene and the promoter used, by techniques known per se, in particular colorimetric, enzymatic, immunological, fluorescence or selection techniques on an appropriate growth medium.
- the activity of the SCF Met3 ° complex is determined by measuring the level of expression and the stability of the protein encoded by the hybrid sequence and / or by the activity of the transcriptional reporter system; in fact, the hybrid protein encoded by said hybrid sequence advantageously retains the intrinsic properties of each of the two fused elements constituting it.
- the Met4-marker protein is selectively degraded (by adding methionine to the culture medium) by the ubiquitin-proteasome pathway (property of the Met4 protein) and can be viewed, in accordance with the associated marker: when the marker present in the hybrid protein is the GFP protein, then the activity of SCF Met30 complex is visualized by observing and quantifying the fluorescence of the hybrid protein GFP-Met4 or GFP-Met30; when the marker present in the hybrid protein is the hemagglutinin (Ha) antigenic motif, then the activity of the SCF Met3 ° complex is visualized by immunological techniques, such as protein transfer techniques (Western Blotting), ELIS techniques A or immunoprecipitation techniques (Harlow E.
- immunological techniques such as protein transfer techniques (Western Blotting), ELIS techniques A or immunoprecipitation techniques (Harlow E.
- the activity of the SCF Met3 ° complex is visualized by growing the yeast on a minimum medium containing no histidine, in the presence of aminotriazole, at concentrations of 0.5 mM to 200 mM. Due to the existence of numerous subunits common to the different SCF complexes, said subunits being in stoichiometric equilibrium, the SCF Met3 ° complex can serve as a model for screening molecules for their ability to modulate either the activity of pathways signaling controlled by all SCF complexes, that of the ubiquitin-proteasome pathway, protein degradation pathway.
- this second system (ubiquitin-proteasome pathway), as implemented in the present invention, using the Met4 protein as substrate, is particularly advantageous for screening molecules or agents capable of acting on pathologies linked to dysfunction of the ubiquitin-proteasome cascade in humans such as cancers, genetic diseases, Parkinson's disease, Alzheimer's disease, inflammatory syndromes and viral infections: simplicity: the induction of the SC3 Met3 ° / Met4 complex system is carried out simply by adding methionine to the growth medium, and when the marker is the GFP protein, the activity of the SCF Me 30 complex is viewed directly by the observation and quantification of the fluorescence emitted, - rapidity of development: reverse genetic techniques, full knowledge of the yeast genome and molecular biology methods adapted to this organism, ensure rapid implementation of stable indicator strains ,
- yeast is a microorganism with rapid growth and high yield, which allows the production of modified cells for a high number of screens,
- yeast is a microorganism whose cultivation, storage, characterization are inexpensive.
- the screening methods according to the invention can in particular be used to select active agents such as anticancer, anti-inflammatory, antiviral or active agents in genetic diseases, in particular in Parkinson's disease and Alzheimer's disease.
- the term “compound or agent” is understood to mean any molecule resulting from synthesis processes or from natural resources.
- the present invention also relates to the use of agents selected by the methods according to the present invention, for the preparation of medicaments intended for the treatment of diseases linked to disorders of the activity of the SCF complexes or of the ubiquitin pathway.
- proteasome such as cancers, genetic diseases, Parkinson's disease, Alzheimer's disease, inflammatory syndromes and viral infections.
- the present invention also relates to plasmids, characterized in that they contain a hybrid sequence, comprising a sequence coding for a protein Met4, Met28 or Met30 in its wild or mutated form, fused in phase with at least one sequence coding for a marker chosen according to the Met protein, from the group consisting of: antigenic peptides, intrinsically fluorescent proteins and proteins with measurable enzymatic activity, said hybrid sequence which can be expressed in yeast, under the control of a promoter, constitutive or inducible.
- a hybrid sequence comprising a sequence coding for a protein Met4, Met28 or Met30 in its wild or mutated form, fused in phase with at least one sequence coding for a marker chosen according to the Met protein, from the group consisting of: antigenic peptides, intrinsically fluorescent proteins and proteins with measurable enzymatic activity, said hybrid sequence which can be expressed in yeast, under the control of a promoter, constitutive or inducible.
- said marker is chosen from antigenic peptides, proteins with intrinsic fluorescence,
- said marker is chosen from antigenic peptides, proteins with intrinsic fluorescence and proteins with measurable enzymatic activity, and
- said marker is chosen from proteins with intrinsic fluorescence and proteins with measurable enzymatic activity.
- said plasmids are selected from the group consisting of:
- Met4, Met28 or Met30 fused to a GFP protein, said sequence being expressed respectively under the control of the constitutive promoters MET4, MET28 or MET30 or under the control of the inducible promoter GAL1,
- HA hemagglutinin
- the subject of the present invention is also plasmids, characterized in that they contain a hybrid sequence, comprising a sequence coding for a Met4 protein, in its wild or mutated form, fused in phase with at least one sequence coding for the factor of binding to LexA DNA, and the TRP1 gene or the LEU2 gene as selection genes for yeasts modified by said plasmids,
- the present invention also relates to plasmids containing a reporter transcription system, consisting of a reporter gene placed under the control of either an appropriate yeast promoter or an appropriate operating sequence, as defined above.
- said plasmids contain the LacZ or XylE reporter gene, expressed under the control of either the MET 16 promoter or LexA operators.
- the present invention also relates to yeast strains, characterized in that they are modified, in a stable manner, by at least one plasmid according to the present invention.
- FIG. 1 illustrates the influence of the addition of methionine at repressive concentrations in the culture medium
- the cells comprising a plasmid coding for the proteins marked by hemagglutinin antigenic motifs (Ha) under the control of GAL1 promoter, and prepared according to the procedure described in Examples 2 and 4, are cultured, according to the procedure described in Example 12, in a minimum medium containing 2% galactose for 90 minutes, either in the presence of methionine at repressive concentration (+ Met), ie in the absence of methionine (- Met);
- B and (C) the total RNAs are extracted, according to the procedure described in Example 12, from cells used in (A) and expressing either the Ha-Met4 hybrid protein or the Ha- hybrid protein Met28.
- the cells are cultured under the conditions described in (A) and are analyzed with MET4, MET16, MET25, MET28.
- “Met4 ⁇ ” corresponds to a W303 cell modified by a chromosomal copy of the inactivated MET4 gene
- “Met28 ⁇ ” corresponds to a W303 cell modified by a chromosomal copy of the inactivated MET2S gene.
- - Figure 2 illustrates the location of the hybrid proteins GFP-Met4 and GFP-Met28 in wild cells, in the absence of methionine (- Met) or in the presence of methionine at a repressive concentration (+ Met).
- the cells are cultivated under the conditions described in Example 12.
- “Hoe” corresponds to the specific indicator for the nuclei, Hoechst 333-42;
- “Name” corresponds to the image obtained by interference microscopy according to Nomarski.
- FIG. 3 illustrates (A) the location of the GFP-Met30 hybrid protein in wild cells, in the absence of methionine (- Met) or in the presence of methionine at a repressive concentration (+ Met).
- the cells are cultured under the conditions described in Example 12.
- "Hoe” corresponds to the Hoechst 333-42 colored indicator;
- “Name” corresponds to the image obtained by interference microscopy according to Nomarski;
- B stability of the Ha-Met30 and Ha-Met30 ⁇ F hybrid proteins in wild W303-1A strains.
- FIG. 4 illustrates the activity of the reporter gene LexAopXylE in the absence of methionine (- Met) or in the presence of methionine at a repressive concentration, from a yeast strain (Cl 90) expressing the hybrid protein coded by pLexMet4-7; visualization is carried out by measuring catechol oxidase.
- Example 1 Construction of the basic plasmids pGal316Flu, pGaI306Flu and pFL39Flu.
- the fragment obtained is digested with the restriction enzymes HindIII and EcoRI and inserted:
- a double-stranded DNA fragment corresponding to the sequence SEQ ID No. 3 and coding for a repeat of 3 hemagglutinin antigenic motifs (Ha) is inserted: (i) in the plasmid pGal316 obtained in point 1.1.1, previously digested with l restriction enzyme EcoRI, the ends of which were made blunt by treatment with the Klenow fragment of DNA polymerase from E. coli, producing the plasmid pGal316Flu,
- the EcoRI site present in the polylinker of the shuttle plasmid S. cerevisiae - E. coli pFL39 has been destroyed.
- the plasmid pFL39 the sequence of which is that deposited in the ⁇ MBL database, with the identifier "CVPFL39", under the number X70483, was digested with EcoRI, the ends made blunt by treatment with the fragment of Klenow of E. DNA polymerase. coli, and the product thus treated was religated on itself producing the plasmid pFL39 ⁇ 0.
- the Ht / idlII-Estl fragment of the plasmid pGal316Flu prepared according to the procedure described in point 1.1. and comprising the promoter region GAL1 and the fragment coding for the antigenic region ⁇ a, was inserted into the plasmid pFL39 ⁇ 0 previously digested with the enzymes HindIII and Pstl.
- Example 2 Plasmids allowing expression in yeast of Ha-Met4 hybrid proteins under the control of the GAL1 promoter.
- the following plasmids allow the expression in yeast S. cerevisiae of derivatives of the Met4 protein comprising a repeat of three hemagglutinin (Ha) antigenic units at their amino terminal end.
- the EcoRl-Xbal DNA fragment of the vector pLexM4 ⁇ 12 (Kuras L. et al, (1995), Mol. Cell. Biol, 15, 208-216) coding for a derivative comprising amino acids 15 to 79 and 180 to 666 of the Met4 protein was cloned into the plasmid pGal316Flu previously digested with the enzymes EcoRI and Xbal, producing the plasmid pGal316FluMet4 ⁇ 12.
- This plasmid can replicate autonomously in yeast.
- the EcoRl-Xbal DNA fragment of the vector pLexM4 ⁇ 37 (Kuras L. et al, (1995), cited reference) coding for a derivative comprising amino acids 15 to 344 and 366 to 666 of the Met4 protein was cloned into the plasmid pGal316Flu previously digested with EcoRI and Xbal enzymes, producing the plasmid pGal316FluMet4 ⁇ 37.
- This plasmid can replicate autonomously in yeast.
- Met4 was cloned into the plasmid pGal316Flu previously digested with enzymes
- the following plasmids allow expression in yeast S. cerevisiae of derivatives of the Met4 protein fused to the green fluorescent protein of Aequora Victoria (Green Fluorescent Protein or GFP). 3.1. Construction of the plasmid pGFPMet4
- a fragment of 710 base pairs (bp) from the plasmid pGFPmut3, coding for the protein GFP3 (Green Fluorescent Protein mut3, product of the GFP gene from Aequora Victoria whose sequence is deposited in the EMBL bank, with the identifier "AVU73901" , under No. U73901) was amplified by PCR (polymerase chain reaction) using the oligonucleotides “olGFPM4-5” of sequence SEQ ID No. 4: 5 'ACGCGAATTCATGTCTAAAGGTGAATTA 3' and “olGFPM4-3” of sequence SEQ ID N ° 5
- the fragment obtained was digested with the restriction enzyme EcoRI and inserted into the plasmid pM4-5 (Kuras L. et al, (1995) reference cited) previously digested with the enzyme EcoRI, producing the plasmid pGFPMet4.
- This plasmid allows expression under the control of the MET4 promoter of a GFP-Met4 hybrid protein comprising amino acids 15 to 666 of Met4, and can replicate autonomously in yeast.
- the EcoRI-EcoRI DNA fragment of the vector pGal316GFPMet4 prepared according to the procedure described in point 3.2 and coding for GFP was cloned in phase in the plasmid pGal316FluMet4 ⁇ 30 previously digested with the enzyme EcoRI, producing the plasmid pGa! 316GFPMet4 ⁇ 30.
- This plasmid can replicate autonomously in yeast.
- the EcoRI-EcoRI DNA fragment of the vector pGal316GFPMet4 prepared according to the procedure described in point 3.2 and coding for GFP was cloned in phase in the plasmid pGal316FluMet4 ⁇ 37 previously digested with the enzyme EcoRI, producing the plasmid pGal316GFPMet4 ⁇ 37.
- This plasmid can replicate autonomously in yeast.
- the DNA fragment Not1-Asp718 of the vector pGal316GFPMet4 prepared according to the procedure described in point 3.2 and comprising all of the GAL1 promoter included in the sequence deposited in the ⁇ MBL database, under the identifier “SCGAL10”, under the n ° K02115 and the GFP-Met4 merger (residues 15 to 666 of Met4), was inserted into the plasmid pRS306 previously digested with the enzymes NotI -Asp718, producing the plasmid pGa! 306GFPMet4. 3.7. Construction of the plasmid pGa! 306GFPMet4 ⁇ 12: The DNA fragment NotI -Asp718 of the vector pGal316GFPMet4 ⁇ 12 prepared according to the procedure described in point 3.3 and comprising all of the
- Example 4 Construction of the plasmids allowing the expression in yeast of hybrid proteins Ha-Met28 and GFP-Met28.
- the EcoRI-BglII DNA fragment of the vector pLexM28-2 (Kuras L. et al, (1996), cited reference) coding for amino acids 1 to 166 of the Met28 protein was cloned into the plasmid pFL39Flu previously digested with EcoRI and BgHl enzymes, producing the plasmid pFL39FluMet28.
- This plasmid can replicate autonomously in yeast. It allows the expression in yeast of a whole Met28 protein comprising at its amino-terminal end a repeat of 3 hemagglutinin (Ha) antigenic units.
- the Ha-Met28 hybrid protein is expressed under the control of the GAL1 promoter.
- the Xbal-Hpal DNA fragment of the vector pMet28-1 (Kuras L. et al, (1996), cited reference) containing the MET28 gene, the sequence of which is that deposited in the ⁇ MBL database, under the identifier “ SC17015 ", under No. U 17015 and its promoter region has been isolated, its ends have been made blunt by treatment with the Klenow fragment of DNA polymerase E. coli and this DNA fragment was inserted into the plasmid pRS314 (Sikorski R. S. et al, (1989), Genetics, 122, 19-27) previously digested with the enzyme SmaI, producing the plasmid p314Met28.
- This plasmid can replicate autonomously in yeast.
- the fragment obtained after PCR and the plasmid p314Met28, previously digested with the enzyme Bglll, were cotransformed in the yeast strain W303-1A (Bailis AM et al, (1990), cited reference), and the prototypic clones for tryptophan were selected.
- the plasmid DNA contained in these clones was extracted, transformed into E. coli and the recombinant plasmid p314Met28GFP identified and characterized by enzymatic digestions and sequence.
- This plasmid allows the expression of a whole Met28 protein fused at its carboxy-terminal end with the GFP protein.
- the Met28-GFP hybrid protein is expressed under the control of the MET28 promoter.
- This plasmid can replicate autonomously in yeast.
- Example 5 Construction of the plasmids allowing the expression in yeast of hybrid proteins Ha-Met30 and GFP-Met30.
- plasmid pGal316FluMet30 ⁇ N previously digested with the enzyme EcoRI, producing the plasmid pGal316FluMet30.
- This plasmid can replicate autonomously in yeast. It allows the expression in yeast of a whole Met30 protein comprising at its amino-terminal end a repeat of 3 anti- hemagglutinin (Ha) genes.
- the Ha-Met30 hybrid protein is expressed under the control of the GAL1 promoter.
- the Eco Rl-Bglll DNA fragment of the vector pGadMet30-1 (Thomas D. et al, (1995), cited reference) coding for amino acids 158 to 640 of the protein Met30 was cloned into the plasmid pFL39Flu previously digested with the enzymes EcoRI and BamEl, producing the plasmid pFL39FluMet30 ⁇ N.
- This plasmid can replicate autonomously in yeast. It allows the expression in yeast of a protein of a Met30 protein truncated from its amino-terminal part but comprising at this end a repetition of 3 hemagglutinin (Ha) antigenic motifs.
- the Ha-Met30 hybrid protein is expressed under the control of the GAL1 promoter.
- This plasmid is constructed according to the procedure described in point 5.3, but expresses a mutated Met30 protein which does not include amino acids 187 to 201.
- the EcoRI-EcoRI DNA fragment of the vector pLexMet30-4 (Thomas D. et al, (1995), cited reference) coding for the first 157 amino acids of the Met30 protein was cloned into the plasmid pFL39FluMet30 ⁇ N previously digested with EcoRI enzyme, producing the plasmid pFL39FluMet30.
- This plasmid can replicate autonomously in yeast. It allows the expression in yeast of a whole Met30 protein comprising at its amino-terminal end a repeat of 3 hemagglutinin (Ha) antigenic units.
- the Ha-Met30 hybrid protein is expressed under the control of the GAL1 promoter.
- a fragment of 710 base pairs (bp) of the plasmid pGFPmut3 coding for the protein GFP3 was amplified by PCR using the oligonucleotides "olM30GFP5" of S ⁇ Q ID N ° 8: 5 'GGGTGCGTAAAAATGTACAAATTCGATCTCAATGATTCT
- the fragment obtained was inserted into the plasmid pGal316FluMet30 by the “gap repair” method (Orr Weaver et a, 1983; reference already cited).
- the fragment obtained after PCR and the plasmid pGal316FluMet30, previously digested with the enzyme Xba / were cotransformed in the strain W303-1A of yeast and the prototrophic clones for uracil were selected.
- the plasmid DNA contained in these clones was extracted, transformed into E. coli and the recombinant plasmid p316FluMet30GFP identified and characterized by enzymatic digestions and sequence. This plasmid allows the expression of a protein
- Ha-Met30 whole fused at its carboxy-terminal end, with the GFP protein and at its amino-terminal end with a repeat of 3 hemagglutinin (Ha) antigenic units.
- the Ha-Met30-GFP hybrid protein is expressed under the control of the GAL 1 promoter. This plasmid can replicate autonomously in yeast.
- the Xbal-SaR DNA fragment of the vector pMet30-1 (Thomas D. et al, (1995), cited reference) containing the MET30 gene, the sequence of which is that deposited in the ⁇ MBL database, under the identifier "SCM ⁇ T30A", under the number L26505 and its promoter region was isolated and inserted into the plasmid pRS316 previously digested with the enzymes Xbal and Sali, producing the plasmid pMet30-8.
- This plasmid can replicate autonomously in yeast.
- the plasmid DNA contained in these clones was extracted, transformed into E. coli and the recombinant plasmid pMet30GFP identified and characterized by enzymatic digestions and sequence.
- This plasmid allows the expression of a whole Met30 protein fused at its carboxy-terminal end, with the GFP protein.
- the Met30-GFP hybrid protein is expressed under the control of the MET30 promoter. This plasmid can replicate autonomously in yeast.
- Example 6 Construction of the plasmids pYi39XyLex, pYi44XyLex, pLexM4-7, pLexM4 ⁇ 239, pM16Zl and pM16Xyll making it possible to visualize the transcriptional activity of the yeast MET genes.
- 6.1 Construction of the plasmid pYi39XvLex:
- this plasmid into the yeast genome at the TRP1 locus can be directed by linearizing this plasmid with the enzyme Stwl and transforming, by the plasmid thus linearized, a yeast strain carrying a point mutation in the trpl gene. 6.1.1. construction of the plasmid pXylex:
- the vector pFL39 was digested with the enzyme Clal and was religated on itself and a plasmid having lost the Clal-Clal fragment containing the original origin of replication ars-cen was selected, producing the vector pYi39. 6.1.3. construction of the plasmid pYi39Xylex:
- the vector pFL44 was digested with the enzyme Clal and was religated on itself and a plasmid having lost the Clal-Clal fragment containing the original 2 ⁇ m replication origin was selected, producing the vector pYi44.
- the BamHI-Pst1 DNA fragment of the vector pXylex prepared according to the procedure described in point 6.1.1 and containing the XylE gene preceded by the promoter region GAL1 -LexA was isolated and inserted into the plasmid pYi44 previously digested with the enzymes BamHI and Pstl, producing the plasmid pYi44Xyl-Lex.
- the hybrid protein thus synthesized is capable of binding to DNA regions comprising LexA operators.
- Met4 was cloned into the plasmid pBTM116 (Margottin M. et al, (1998), cited reference) previously digested with the enzymes EcoRI and BamHI, producing the plasmid pLexM4 ⁇ 239. This plasmid can replicate autonomously in yeast.
- This plasmid can replicate autonomously in yeast and allows the expression of E. coli ⁇ -galactosidase under the control of the MET16 promoter.
- the DNA fragment containing the promoter of the MET16 gene (comprising the nucleotides -535 to -1, numbered from the initiation codon of the MET16 gene) was amplified by PCR from the plasmid pM16-1 (Thomas D. and al. (1990), J. Biol. Chem., 265, 15518-15524) using the oligonucleotides “M16OL5” of sequence S ⁇ Q ID No. 16
- this plasmid into the yeast genome at the LEU2 locus can be directed by linearizing this plasmid with the enzyme Asp7l8 and transforming, by the plasmid thus linearized, a yeast strain carrying a point mutation in the leu2 gene.
- this plasmid makes it possible to obtain a modified yeast strain, stably expressing the catechol oxidase of P. putida, placed under the control of the MET 16 gene.
- Example 7 Yeast strains expressing a hybrid protein GFP-Met4.
- strains carry at the level of the locus URA3 (chromosome V, left arm), an artificial gene allowing the expression of a Met4 protein, modified or not, under the control of the promoter of the GAL1 gene.
- URA3 can be directed by linearizing this plasmid with the enzyme Stul and transforming, by the plasmid thus linearized, a yeast strain carrying a point mutation in the ura3 gene.
- the GFP-Met4 fusion thus integrated into the chromosomal locus URA3 is expressed under the control of the promoter GAL1.
- the strains are transformed according to the procedure described in point 7.1. from the plasmid pGal306GFPMet4 ⁇ 12.
- strains are transformed according to the procedure described in point 7.1. from the plasmid pGal306GFPMet4 ⁇ 30. 7.4. Preparation of strains from pGa! 306GFPMet4 ⁇ 37:
- the strains are transformed according to the procedure described in point 7.1. from the plasmid pGal306GFPMet4 ⁇ 37.
- Example 8 Yeast strains carrying the XyLE gene coding for catechol oxidase under the control of operators LexA.
- strains carry at the URA3 locus (chromosome V, left arm) or at the TRP1 locus (chromosome IV, right arm) an artificial gene comprising the XylE gene from Pseudomonas putida under the control of operators LexA.
- the expression of this gene is directed by the hybrid protein LexA-Met4 expressed from the replicative plasmid pLexM4-7.
- strains which have been prepared are collated in Table II, below.
- URA3 can be directed by linearizing this plasmid with the enzyme Stwl and transforming by the plasmid thus linearized, a yeast strain carrying a point mutation in the ura3 gene (Orr-Weaver TL et al. (1983) and Rothstein R. (1991) ), references cited).
- TRP1 can be directed by linearizing this plasmid with the enzyme Stwl and transforming by the plasmid thus linearized, a yeast strain carrying a point mutation of the trpl gene (Orr-Weaver TL et al. (1983) and Rothstein R. (1991), references cited).
- Example 9 Yeast strain carrying the XylE gene coding for catechol oxidase under the control of the promoter of the MET25 gene. 9.1. Preparation of strain CC634-2D:
- This strain is derived from the cross between the CI2-11D strain (Jacquemin-Faure I. et al, (1994), reference cited) and the CD 106 strain (Thomas D. et al, (1992), cited reference). It includes, integrated into the LEU2 locus (chromosome III, left arm) a XylE gene placed under the control of the promoter of the MET25 gene. 9.2. Characteristics of the CC634-2D strain:
- the genotype of this strain is as follows: Mata, ade2, his3, trpl, ura3, met4 :: TRPl, leu2 :: proMet25-XylE :: LEU2.
- Example 10 Yeast strain carrying the HIS3 gene under the control of LexA operators. These strains carry at the LYS2 locus (chromosome II, right arm) an artificial gene comprising the HIS3 gene of S. cerevisiae placed under the control of operators LexA. The expression of this gene is directed by the hybrid protein LexA-Met4 expressed from the replicative plasmid pLexM4-7.
- This strain comes from the cross between the L40 strain ( ⁇ ollenberg SM et al, (1995), Mol. Cell Biol, 15, 3813-3822) and the CC816-1D strain of Mat genotype, ade2, leu2, lys2, trpl, ura3 .
- the genotype of this strain is as follows: Mata, ade2, his3, leu2, trpl, ura3, lys2 :: LexAop-HIS3 :: LYS2 10.2.2. Characteristics of the CY25-1D strain: The genotype of this strain is as follows: Mata, ade2, his3, leu2, trpl, ura3 met32 :: URA3, lys2 :: LexAop-HIS3 :: LYS2
- Example 11 Yeast strain carrying the LacZ gene under the control of LexA operators.
- strains carry at the URA3 locus (chromosome V, left arm) an artificial gene comprising the LacZ gene of E. coli under the control of LexA operators.
- the expression of this gene is directed by the hybrid protein LexA-Met4 expressed from the replicative plasmid pLexM4-7. 1 1.1. Preparation of strain CC801-3B
- This strain is derived from the cross between the L40 strain (Hollenberg S. M. et al, (1995), reference cited) and the CD 106 strain (Thomas D. et al, (1992), cited reference).
- strain CC801-3B The genotype of this strain is as follows: Mata, ade2, his3, leu2, trpl, ura3 met4 :: TRPl, ura3 :: LexAop-LacZ :: URA3
- Example 12 Determination of the level of expression and the stability of the proteins expressed from the hybrid sequences contained in the plasmids according to the invention. 12.1. Procedure:
- Yeast cells comprising a plasmid coding for the proteins labeled by Ha, under the control of the GAL1 promoter, are cultured in a medium containing raffinose.
- the induction of the hybrid protein, expressed under the control of the GAL1 promoter, is carried out for 2 to 10 hours, by transferring the cells to a medium containing 2 to 5% of galactose.
- Yeast cells comprising a plasmid coding for the proteins labeled with the GFP protein, under the control of the GAL1 promoter, are cultured in a medium containing raffinose and the induction of the hybrid protein is carried out according to the procedure described above.
- the activity of the LexAopXyle reporter gene is measured in a yeast strain (Cl 90) expressing the hybrid protein encoded by the plamide pLexMet4-7 and cultured under the conditions described above.
- the activity is measured by a visualization technique based on the measurement of catechol oxidase according to conventional techniques.
- the protein Ha-Met4 has a half-life of the order of 20 minutes in the absence of methionine (FIG. 1A, - Met) and a half-life of the order of 5 minutes under repressive conditions, it is ie in the presence of methionine (Figure 1A, + Met).
- the Ha-Met28 protein has a half-life of the order of 20 minutes in the absence of methionine ( Figure 1A, - Met).
- the repressive conditions do not modify the half-life of the protein Ha-Met28 (FIG. 1A, + Met).
- the Ha-Met30 protein has a half-life of the order of 20 minutes in the absence of methionine (FIG. 3B, - Met).
- the Ha-Met30 ⁇ F hybrid protein appears less stable than the Ha-Met30 hybrid protein, but repressive conditions do not reduce the stability of this hybrid protein. b) Y. discounting . by . prot . undertake hybri
- the GFP-Met4 hybrid protein is located in the nucleus when cells are grown in the absence of methionine.
- the GFP-Met28 hybrid protein is always present in the nucleus, whether in the absence or in the presence of methionine ( Figure 2B).
- the GFP-Met30 hybrid protein is always present in the nucleus, whether in the absence or in the presence of methionine ( Figure 3A).
- the cells In the absence of methionine, the cells are colored yellow, proof that the protein is expressed. In the presence of methionine, the cells are white due to repression.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Neurology (AREA)
- Immunology (AREA)
- Neurosurgery (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Hospice & Palliative Care (AREA)
- Psychology (AREA)
- Psychiatry (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2001542563A JP2003517307A (ja) | 1999-12-01 | 2000-11-30 | ユビキチン−リガーゼscf複合体の活性化を調節し得る化合物の細胞スクリーニング方法及びその使用 |
DE60039452T DE60039452D1 (de) | 1999-12-01 | 2000-11-30 | Zelluläres testverfahren für zusammensetzungen, die die aktivität des ubiquitin-ligase scf komplexes beeinflussen und dessen anwendungen |
CA002393115A CA2393115A1 (fr) | 1999-12-01 | 2000-11-30 | Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications |
EP00985364A EP1234041B1 (fr) | 1999-12-01 | 2000-11-30 | Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications |
US11/749,933 US20080199864A1 (en) | 1999-12-01 | 2007-05-17 | Methods for cell screening of compounds capable of modulating the activity of ubiquitin-ligase scf complexes and their uses |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR99/15138 | 1999-12-01 | ||
FR9915138A FR2801902B1 (fr) | 1999-12-01 | 1999-12-01 | Procedes de criblages fonctionnels d'agents aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/749,933 Continuation US20080199864A1 (en) | 1999-12-01 | 2007-05-17 | Methods for cell screening of compounds capable of modulating the activity of ubiquitin-ligase scf complexes and their uses |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001040499A2 true WO2001040499A2 (fr) | 2001-06-07 |
WO2001040499A3 WO2001040499A3 (fr) | 2001-10-25 |
Family
ID=9552767
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2000/003342 WO2001040499A2 (fr) | 1999-12-01 | 2000-11-30 | Procedes de criblage cellulaire de composes aptes a moduler l'activite des complexes ubiquitine-ligases scf et leurs applications |
Country Status (8)
Country | Link |
---|---|
US (2) | US20030175679A1 (fr) |
EP (1) | EP1234041B1 (fr) |
JP (1) | JP2003517307A (fr) |
AT (1) | ATE400659T1 (fr) |
CA (1) | CA2393115A1 (fr) |
DE (1) | DE60039452D1 (fr) |
FR (1) | FR2801902B1 (fr) |
WO (1) | WO2001040499A2 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002042442A2 (fr) * | 2000-11-24 | 2002-05-30 | Biotecon Diagnostics Gmbh | Nouvelle souche de levure destinee a l'alimentation |
DE10230224A1 (de) * | 2002-07-04 | 2004-04-01 | Axaron Bioscience Ag | Verfahren zur Produktion von S-Adenosyl-L-Methionin durch Fermentation von genetisch modifizierten Mikroorganismen |
FR2859733A1 (fr) * | 2003-09-15 | 2005-03-18 | Centre Nat Rech Scient | Procede de criblage fonctionnel d'agents aptes a moduler l'activite des ubiquitine hydrolases et leurs applications |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7608682B2 (en) * | 2002-10-30 | 2009-10-27 | Meso Scale Technologies Llc | Substrates of N-end rule ubiquitylation and methods for measuring the ubiquitylation of these substrates |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997012962A1 (fr) * | 1995-10-04 | 1997-04-10 | Cold Spring Harbor Laboratory | Ubiquitine ligases et utilisations associees |
WO1999004033A1 (fr) * | 1997-07-16 | 1999-01-28 | Mitotix, Inc. | ESSAI PERMETTANT DE DECELER LES INHIBITEURS DE LA PROTEOLYSE DU I KAPPA B (IλB) |
WO1999018989A1 (fr) * | 1997-10-16 | 1999-04-22 | Baylor College Of Medicine | Proteines et genes a sequence f |
US5932425A (en) * | 1997-02-18 | 1999-08-03 | Signal Pharmaceuticals, Inc. | Compositions and methods for modulating cellular NF-κB activation |
WO1999038969A1 (fr) * | 1998-01-30 | 1999-08-05 | Institut National De La Sante Et De La Recherche Medicale | PROTEINE HUMAINE β-TrCP |
WO2000022110A2 (fr) * | 1998-10-09 | 2000-04-20 | President And Fellows Of Harvard College | Proteolyse ciblee par recrutement de ligases proteiniques d'ubiquitine |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE207538T1 (de) * | 1995-02-09 | 2001-11-15 | Behringwerke Ag | Verfahren zur produktion von proteinen |
US5885779A (en) * | 1997-09-09 | 1999-03-23 | University Of British Columbia | Repressed trans-activator system for characterization of protein-protein interactions |
-
1999
- 1999-12-01 FR FR9915138A patent/FR2801902B1/fr not_active Expired - Fee Related
-
2000
- 2000-11-30 US US10/148,170 patent/US20030175679A1/en not_active Abandoned
- 2000-11-30 WO PCT/FR2000/003342 patent/WO2001040499A2/fr active IP Right Grant
- 2000-11-30 JP JP2001542563A patent/JP2003517307A/ja not_active Withdrawn
- 2000-11-30 AT AT00985364T patent/ATE400659T1/de not_active IP Right Cessation
- 2000-11-30 EP EP00985364A patent/EP1234041B1/fr not_active Expired - Lifetime
- 2000-11-30 DE DE60039452T patent/DE60039452D1/de not_active Expired - Fee Related
- 2000-11-30 CA CA002393115A patent/CA2393115A1/fr not_active Abandoned
-
2007
- 2007-05-17 US US11/749,933 patent/US20080199864A1/en not_active Abandoned
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997012962A1 (fr) * | 1995-10-04 | 1997-04-10 | Cold Spring Harbor Laboratory | Ubiquitine ligases et utilisations associees |
US5932425A (en) * | 1997-02-18 | 1999-08-03 | Signal Pharmaceuticals, Inc. | Compositions and methods for modulating cellular NF-κB activation |
WO1999004033A1 (fr) * | 1997-07-16 | 1999-01-28 | Mitotix, Inc. | ESSAI PERMETTANT DE DECELER LES INHIBITEURS DE LA PROTEOLYSE DU I KAPPA B (IλB) |
WO1999018989A1 (fr) * | 1997-10-16 | 1999-04-22 | Baylor College Of Medicine | Proteines et genes a sequence f |
WO1999038969A1 (fr) * | 1998-01-30 | 1999-08-05 | Institut National De La Sante Et De La Recherche Medicale | PROTEINE HUMAINE β-TrCP |
WO2000022110A2 (fr) * | 1998-10-09 | 2000-04-20 | President And Fellows Of Harvard College | Proteolyse ciblee par recrutement de ligases proteiniques d'ubiquitine |
Non-Patent Citations (9)
Title |
---|
CIECHANOVER A ET AL: "THE UBIQUITIN-MEDIATED PROTEOLYTIC SYSTEM: INVOLVEMENT OF MOLECULAR CHAPERONES, DEGRADATION OF ONCOPROTEINS, AND ACTIVATION OF TRANSCRIPTIONAL REGULATORS" COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY,US,NEW YORK, NY, vol. 60, 1995, pages 491-501, XP002069094 * |
CORMACK B P ET AL: "FACS-optimized mutants of the green fluorescent protein (GFP)" GENE,NL,ELSEVIER BIOMEDICAL PRESS. AMSTERDAM, vol. 173, no. 1, 1996, pages 33-38, XP004042850 ISSN: 0378-1119 * |
KOLODZIEJ P A ET AL: "EPITOPE TAGGING AND PROTEIN SURVEILLANCE" METHODS IN ENZYMOLOGY,US,ACADEMIC PRESS INC, SAN DIEGO, CA, vol. 194, 1991, pages 508-519, XP002057283 ISSN: 0076-6879 * |
PATTON E E ET AL: "Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis" TRENDS IN GENETICS,NL,ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, vol. 14, no. 6, 1 juin 1998 (1998-06-01), pages 236-243, XP004121083 ISSN: 0168-9525 cité dans la demande * |
PATTON ET AL.: "Cdc53 IS A SCAFFOLD PROTEIN FOR MULTIPLE Cdc34/Skp1/F-box PROTEIN COMPLEXES THAT REGULATE CELL DIVISION AND METHIONINE BIOSYNTHESIS IN YEAST" GENES DEV., vol. 12, 1998, pages 692-705, XP002143736 cité dans la demande * |
PATTON ET AL.: "SCF(Met30)-MEDIATED CONTROL OF THE TRANSCRIPTIONAL ACTIVATOR Met4p IS REQUIRED FOR THE G1-S TRANSITION" EMBO J., vol. 19, no. 7, avril 2000 (2000-04), pages 1613-1624, XP002143738 * |
ROUILLON ET AL.: "FEEDBACK-REGULATED DEGRADATION OF THE TRANSCRIPTIONAL ACTIVATOR Met4 IS TRIGGERED BY THE SCF(Met30) COMPLEX" EMBO J., vol. 19, no. 2, janvier 2000 (2000-01), pages 282-294, XP002143739 * |
SPELLMAN ET AL.: "COMPREHENSIVE IDENTIFICATION OF CELL CYCLE-REGULATED GENES OF THE YEAST SACCHAROMYCES CEREVISIAE BY MICROARRAY HYBRIDIZATION" MOL.BIOL.CELL, vol. 9, décembre 1998 (1998-12), pages 3273-3297, XP002143737 * |
THOMAS AND SURDIN-KERJAN: "METABOLISM OF SULFUR AMINO ACIDS IN SACCHAROMYCES CEREVISIAE" MICROBIOL. AND MOL. BIOL., vol. 61, no. 4, décembre 1997 (1997-12), pages 503-532, XP002143735 cité dans la demande * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002042442A2 (fr) * | 2000-11-24 | 2002-05-30 | Biotecon Diagnostics Gmbh | Nouvelle souche de levure destinee a l'alimentation |
WO2002042442A3 (fr) * | 2000-11-24 | 2002-09-19 | Biotecon Diagnostics Gmbh | Nouvelle souche de levure destinee a l'alimentation |
DE10230224A1 (de) * | 2002-07-04 | 2004-04-01 | Axaron Bioscience Ag | Verfahren zur Produktion von S-Adenosyl-L-Methionin durch Fermentation von genetisch modifizierten Mikroorganismen |
FR2859733A1 (fr) * | 2003-09-15 | 2005-03-18 | Centre Nat Rech Scient | Procede de criblage fonctionnel d'agents aptes a moduler l'activite des ubiquitine hydrolases et leurs applications |
WO2005028613A2 (fr) * | 2003-09-15 | 2005-03-31 | Centre National De La Recherche Scientifique | Procede de criblage fonctionnel d'agents aptes a moduler l'activite des ubiquitine hydrolases et leurs applications. |
WO2005028613A3 (fr) * | 2003-09-15 | 2005-06-16 | Centre Nat Rech Scient | Procede de criblage fonctionnel d'agents aptes a moduler l'activite des ubiquitine hydrolases et leurs applications. |
Also Published As
Publication number | Publication date |
---|---|
EP1234041B1 (fr) | 2008-07-09 |
WO2001040499A3 (fr) | 2001-10-25 |
EP1234041A2 (fr) | 2002-08-28 |
DE60039452D1 (de) | 2008-08-21 |
FR2801902B1 (fr) | 2005-03-04 |
ATE400659T1 (de) | 2008-07-15 |
FR2801902A1 (fr) | 2001-06-08 |
US20080199864A1 (en) | 2008-08-21 |
CA2393115A1 (fr) | 2001-06-07 |
JP2003517307A (ja) | 2003-05-27 |
US20030175679A1 (en) | 2003-09-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Li et al. | The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity | |
US5466576A (en) | Modulation of PIF-1-type helicases | |
EP1530584B1 (fr) | Peptides synthetiques ou naturels liant la proteine phosphatase 2a, methode d'identification et utilisations | |
Rivero et al. | Rho signaling in Dictyostelium discoideum | |
US20080199864A1 (en) | Methods for cell screening of compounds capable of modulating the activity of ubiquitin-ligase scf complexes and their uses | |
EP1049775B1 (fr) | Proteine humaine btrcp | |
FR2740454A1 (fr) | Peptides capables d'inhiber l'endocytose de l'app et sequences nucleotidiques correspondantes | |
EP0941319A1 (fr) | Peptides capables d'inhiber l'endocytose de l'app et sequences nucleotidiques correspondantes | |
KR100445912B1 (ko) | 단백질간의 상호 결합을 조사할 수 있는 이중 라이브러리단백질 결합 검색방법에 의한 검색 방법 및 이에 이용되는재조합 효모 균주 | |
EP0977840A2 (fr) | Methodes de detection d'interactions entre plusieurs proteines | |
EP1003844B1 (fr) | Kinase activatrice des proteines-kinases cycline dependantes, et ses utilisations | |
Raina | Regulation of meiotic prophase checkpoint function by the AAA+ ATPase Pch2 and the HORMA protein Hop1 | |
WO2005028613A2 (fr) | Procede de criblage fonctionnel d'agents aptes a moduler l'activite des ubiquitine hydrolases et leurs applications. | |
Singh | Studies on the Sexual Development of Aspergillus Nidulans | |
Lanteri | Investigating the interplay between DNA replication and meiotic recombination | |
Piña | Functional Analyses of HPH1and HPH2 Revealed Roles in Post-Translational Protein Translocation and Nutrient Translocation and Nutrient Signaling | |
EP1730297A1 (fr) | Procede de criblage d'agents modulant l'ubiquitination de la proteine lkb alpha et moyens destines a la mise en oeuvre dudit procede | |
Maleri | Ste7 variants that promote pathway specificity in S. cerevisiae | |
Maleri | Ste7 variants that promote pathway specificity in Saccharomyces cerevisiae | |
EP1941281A1 (fr) | Procede de criblage d'agents modulant l'activite du proteasome et moyens destines a la mise en oeuvre dudit procede | |
EP1144651A1 (fr) | Polypeptides derives de jnk3 | |
EP1641919A2 (fr) | Applications d une nouvelle classe d' enzymes : les sulf iredoxines | |
Cerveny | The molecular mechanisms of mitochondrial division | |
EP1257642A1 (fr) | Partenaires du domaine ptb1 de fe65, preparation et utilisations | |
WO2000032768A1 (fr) | Polypeptides capables d'interagir avec la topoisomerase iii alpha humaine |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2001 542563 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2393115 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000985364 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2000985364 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10148170 Country of ref document: US |
|
WWG | Wipo information: grant in national office |
Ref document number: 2000985364 Country of ref document: EP |